Soybeans and other plant parts contain large amounts of lipoxygenases which accelerate the oxidation of unsaturated fatty acids contained therein. The term lipoxygenase (linoleate:oxygen oxidoreductase, EC 1,13,11,12), also known as lipoxidase and carotene oxidase, refers to a class of dioxygenases that catalyze the peroxidation of molecules containing cis,cis-1,4-pentadiene moieties. Lipoxygenases (LOXs) are essentially ubiquitous among eukaryotic organisms and have been demonstrated to exist in many tissues of numerous higher plants and animals. Multiple LOX forms or isozymes are often present in tissues. For example, soybean seeds contain at least three LOX isozymes which are encoded by distinct genes. These genes have been cloned and sequenced. They have regions of high homology. A LOX gene cloned from human leukocytes suggests a common evolutionary origin of plant and animal LOXs (see, Hildebrand et al. 1988 and references therein).
The principal substrates for LOXs in higher plants are linoleic acid (18C long and 2 double bonds, 1 of which is positioned 6C in from the terminal (methyl) or omega (omega)end=C18:2,omega 6) and .alpha.-linolenic acid (C18:3,omega 3). These are the terminal fatty acids synthesized in most plant tissues. The first principal fatty acid produced in fatty acid biosynthesis is palmitic acid (C16:0) which can be elongated to stearic acid (C18:0), which can be desaturated to oleic acid (C18:1). In plants, fatty acid biosynthesis up to oleic acid occurs in the plastids. Subsequent desaturation of oleic acid to linoleic and linolenic acid can occur in the chloroplasts as well as the endoplasmic reticulum (Wang and Hildebrand, "Biosynthesis and Regulation of Linolenic Acid in Higher Plants", Plant Physiol. Biochem. 26 (1988)). Linolenic acid is the most abundant fatty acid in most plant tissues whereas linoleic acid is often the most abundant fatty acid in plant seeds. Linoleic acid is the precursor of other omega 6 fatty acids such as gamma-linolenic acid (6,9,12 all cis C18:3,omega 6) and arachidonic acid (C20:4) and .alpha.-linolenic acid is the precursor of other omega 3 fatty acids such as eicosapentaenoic (C20:5) and docosahexaenoic (C22:6) acids (all of which are substrates for LOX) in the food chain.
Much of the interest in LOX is due to the importance of this enzyme in the post-harvest physiology of many food products. For many seeds with high levels of LOX and linoleic acid such as soybean seeds, the production of hexanal represents a particular problem. Hexanal, even when present in foods in very low concentrations (e.g., 5 ppb), has highly undesirable odor and flavor creating great difficulties in the production of acceptable food products containing for example, soybean homogenates. Additionally, the destruction of LOX activity is thought to be one of the principal reasons for the need for blanching of food products prior to freezing. LOX is also thought to be important in the formation of the flavor components of many fruits and vegetables including cucumbers, tomatoes, melons, etc.
Four major enzyme systems are operative in higher plants by which fatty acids are oxidatively modified: .alpha.-oxidation, .beta.-oxidation, omega-oxidation and the LOX pathway. LOX catalyzes the peroxidative modification of polyunsaturated fatty acids which leads to the formation of various secondary lipid oxidation. This is shown in the reaction scheme set forth in FIG. 3.
The lipoxygenase (LOX) pathway can be summarized as follows. The initial event is thought to be the release of free fatty acids by lipases, but it is not known that this is always necessary. Linoleic and linolenic acids (shown in FIG. 3) are the principal substrates for LOX. The first step of the LOX-catalyzed reaction is the stereospecific removal of hydrogen from the C11 methylene group (step 1). Removal of the 11-pro-S-hydrogen results in a rearrangement to form a free radical at C13 (step 2) and LOX is reduced to the Fe.sup.2+ form. Under aerobic conditions, the LOX-fatty acid radical complex subsequently reacts with O.sub.2 forming a fatty acid peroxy radical (step 3). However, step 3 cannot take place under anaerobic conditions and thus the fatty acid radical is released from the LOX enzyme and alternative reactions occur for some LOX isozymes (see text) (step 3a) at least for the linoleoyl radical and presumably for the linolenyl radical. The fatty acid radicals (formed after steps 1 and 2) and peroxy fatty acid radicals (formed at step 3) are normally bound to the LOX enzyme (intermediates bound to the LOX enzyme are enclosed in brackets). The final step of the LOX-catalyzed reaction is the reduction of the fatty acid peroxy radical to a hydroperoxide and the oxidation of LOX back to the Fe.sup.3+ form and release of the fatty acid product from the enzyme (step 4). The fatty acid hydroperoxides are subsequently metabolized by hydroperoxide lyase to C-6 and C-12 products (step 5) (or C-9 products with some other LOX/lyase systems) or by hydroperoxide dehydrase at least in the case of hydroperoxy-linolenic acid) to 12-oxo-cis, cis-10, 15-phytodienoic acid (12-oxo-PDA) which can be converted ultimately into jasmonic acid.
The initial event in the LOX pathway is thought to be release of free fatty acids from glycerolipids (the vast bulk of fatty acids in living cells is esterified in glycerolipids). However, some LOXs such as the reticulocyte LOX can directly attack phospholipids and even biological membranes (Schewe et al., "Enzymol and Physiology of Reticulocyte Lipoxygenase: comparison with other lipoxygenase", Adv. Enzymol. Related Areas Mol. Bio. 58: 191-272 (1986)).
Typical LOXs contain 1 mol of non-heme iron per mol of enzyme. LOX must be in the oxidized or Fe.sup.3+ form for LOX catalyzed oxidations to proceed. LOX can be activated or oxidized by its own lipid hydroperoxide product (which at high concentrations can also cause the eventual destruction of LOX activity). LOX-Fe.sup.3+ usually binds to either C18:2 or C18:3 in the case of higher plants and catalyzes the stereospecific removal of hydrogen from the C11 methylene group (FIG. 3). Removal of the 11-pro-S-hydrogen and subsequent rearrangement leads to the formation of the C13 radical as shown for soybean LOX-1 (FIG. 3) and LOX is reduced to the Fe.sup.2+ form. Removal of the 11-pro-R-hydrogen, which can occur with soybean LOX 2 and 3 and other LOXs, however, results in the rearrangement of the free radical to the C-9 position (not shown).
Usually the LOX-fatty acid radical complex subsequently reacts with O.sub.2 forming a lipid peroxy radical (see, FIG. 3). However, step 3 cannot take place under anaerobic conditions. Alternative reactions, therefore, take place in the case of many LOXs such as soybean LOX-1 releasing fatty acid radicals from the enzyme (FIG. 3). This reaction occurs with the linoleic acid radical and is presumed to occur with the linolenic acid radical. This leads to the so-called lipo-hydroperoxidase reactions of LOXs yielding fatty acid dimers and oxodienoic acids. For some LOXs, however, such as soybean LOX-3 and a pea LOX, reaction 3a tends to occur aerobically and the fatty acid radicals are released from the LOX enzyme and in this case can react with O.sub.2 yielding peroxy radicals. These peroxy radicals are thought to be responsible for the co-oxidation reactions such as the oxidation of carotenoids and chlorophylls (Schewe et al., (1986)).
The final step of the primary LOX reactions is the reduction of the fatty acid hydroperoxy radical to a hydroperoxide (e.g., 13-(S)-hydroperoxy-9-cis-11-trans-octadecadienoic acid) and the oxidation of LOX back to the Fe.sup.3+ form and release of the fatty acid product from the enzyme (see, FIG. 3, step 4). Hydrogen abstraction is the rate-limiting step of the overall reaction. The decisive feature of all LOX-catalyzed reactions in both plants and animals is the homolytic cleavage of a sigma bond (e.g., C-H) with the formation of intermediate radicals.
The fatty acid hydroperoxides resulting from LOX action are metabolized by one of two major pathways operative in higher plant tissues. In one pathway, hydroperoxide lyase catalyzes the cleavage of 13-hydroperoxy linoleic or linolenic acid into the 12 carbon compound, 12-oxo-9-dodecenoic acid, and the 6 carbon aldehydes, hexanal or cis-3-hexenal (see, FIG. 3 step 5). Nine carbon oxo fatty acids and aldehydes are formed from the 9-hydroperoxy fatty acids. The 12-oxo-cis-9-dodecenoic acid and cis-3-hexenal undergo isomerization to the more stable 12-oxo-trans-10-dodecenoic acid and trans-2-hexenal (see, FIG. 3) (Hatanaka et al., Chem. Phys. Lipids 44: 341-361 (1987); Vick and Zimmerman, "Oxidative Systems for Modification of Fatty Acids: the Lipoxygenase Pathway"-in The Biochem. of Plants: A Comprehensive Treatise, Stumpf. Ed., Vol. 9, pp 53-90, Academic Press, Orlando, Fla. (1987a)). Hexanal and hexenals are often converted to their corresponding alcohols by alcohol dehydrogenase. cis-3-Hexen-1-ol and trans-2-hexenal are known as leaf alcohol and aldehyde and are associated with the "green odor" of leaves as is characteristic for freshly cut grass. The 12-oxo-trans-10-dodecenoic acid, known as wound hormone or traumatin, is readily oxidized non-enzymatically to trans-2-dodecenedioic acid, commonly known as traumatic acid.
The second pathway for metabolism of hydroperoxy fatty acids (at least for linolenic acid) in plants is the conversion into an allene oxide by hydroperoxide dehydrase (Vick and Zimmerman (1987a), supra). The allene oxide can undergo hydrolysis resulting in the formation of ketols. Alternatively, the allene oxide can undergo rearrangement and cyclization resulting in the formation of 12-oxo-cis,cis-10,15-phytodienoic acid (12-oxo-PDA). The 12-oxo-PDA can be converted by a series of reactions into jasmonic acid (Vick and Zimmerman 1987a, supra; Vick and Zimmerman, Plant Physiol. 85: 1073-1078 (1987) (Vick and Zimmerman (1987b))).
In spite of the rapid increase in information concerning the role of LOX in mammalian physiology and of the fairly extensive biochemical and genetic studies that have been done with plant LOXs, little definitive information is available concerning physiological roles for plant LOXs. (Mack et al., Current Topics Biol. Med. Res. 13: 127-154 (1987); Vick and Zimmerman (1987a), supra, and Hildebrand et al. (1988), supra, recently presented reviews on this subject).
Hexanal is a volatile aldehyde formed from soybean seeds during processing. This compound has an undesirable aroma which has limited the widespread use of soybean proteins in food products (Rackis, J. J., Sessa, D. J. & Honig, D. H., J. Amer. Oil Chem. Soc. 56: 262-271 (1979)). Lipoxygenases (linoleate: oxygen oxidoreductase, EC 1.13.11.12) which are thought to be key enzymes responsible for hexanal formation, exist in soybean seeds as three distinct isozymes (Arai, S., et al. Agr. Biol. Chem. 34: 1420-1423 (1970); Wolf, W. J. J. Agr. Food Chem. 23: 136-140 (1975); Axelrod, B., Cheesbrough, T. M. & Laakso, S. Methods Enzymol. 71: 441-451 (1981)).
It was thought that all three lipoxygenase isozymes contribute to hexanal production, with lipoxygenase isozyme 2 being the most effective (Rackis, J. J., Sessa, D. J. & Honig, D. H. J. Amer. Oil Chem. Soc. 56: 262-271 (1979); Arai, S., et al. Agr. Biol. Chem. 34: 1420-1423 (1970); Wolf, W. J. J. Agr. Food Chem. 23: 136-140 (1975); Buttery, R. G., et al. J. Ag. Food Chem. 17: 1322-1327 (1969); Hatanaka, A., Kajiwara, T. & Sekiya, J. Chem. Phys. Lipids 44: 341-361 (1987); Matoba, T. et al. J. Agric. Food Chem. 33: 852-855 (1985)).
Hexanal is a potent odor and flavor compound with a very low olfactory threshold (Buttery, R. G., et al. J. Ag. Food Chem. 17: 1322-1327 (1969)) which makes it undesirable in many food products. It is thought to be formed by hydroperoxidation of linoleic acid (cis,cis-9,12-octadecadienoic acid) in plant tissues through the action of lipoxygenase and subsequent cleavage of the product by hydroperoxide lyase (Hatanaka, A., Kajiwara, T. & Sekya, J. Chem. Phys. Lipids 44: 341-361 (1987)). Soybean (Glycine max L. Merr.) seeds and other plant parts contain relatively high levels of both linoleate and lipoxygenase and high levels of hexanal are produced from aqueous homogenates of soybean seeds (Rackis, J. J., Sessa, D. J. & Honig, D. H. J. Amer. Oil Chem. Soc. 56: 262-271 (1979); Arai, S., et al. Agr. Biol. Chem. 34: 1420-1423 (1970); Wolf, W. J. J. Agr. Food Chem. 23: 136-140 (1975); Axelrod, B., Cheesbrough, T. M. & Laakso, S. Meth. Enzymol. 71: 441-451 (1981)). This has limited the use of whole soybeans and soybean protein as well as other products in many foods.
Soybeans have been screened for mutants missing seed lipoxygenase(s). Mutants with nondetectable or very low lipoxygenase activity were found for all three known soybean seed lipoxygenase isozymes (Hildebrand, D. F. & Hymowitz, T. J. Amer. Oil Chem. Soc. 58: 583-586 (1981); Kitamura, K. J. Agric. Biol. Chem. 48: 2339-2343 (1984)). The null mutants were inherited as simple recessive alleles. Lipoxygenases 1 and 2 were tightly linked but lipoxygenase 3 was inherited independently of lipoxygenases 1 and 2 (Davies, C. S. & Nielsen, N. C. Crop Sci. 26: 460-463 (1986)). Davies and Nielsen (Davies, C. S. & Nielsen, N. C. Crop Sci. 27: 370-371 (1987)) developed near-isogenic lines backcrossed to the soybean cultivar "Century" that are homozygous for one or two of the lipoxygenase null alleles. No triple null lines have been found because of the tight linkage of lipoxygenase 1 and 2 and possible lethality of low frequency recombinants. Matoba et al. (Matoba, T. et al. J. Agric. Food Chem. 33, 852- 855 (1985) examined the generation of hexanal by aqueous seed homogenates of the original lipoxygenase null mutants and concluded that lipoxygenase 2 is largely responsible for the generation of hexanal. Davies et al. (Davies, C. S., Nielsen, S. S. & Nielsen, N. C. J. Amer. Oil Chem. Soc. 64, 1428-1432 (1987)) also concluded that elimination of lipoxygenase 2 through genetic selection was important in the flavor improvement of soybean preparations.
Japanese Patent Application JA-137965 to Sugiyama Sangyo Kag describes the manufacture of noodles having improved texture and color with flour containing a small amount of soybean extract with lipoxidase activity. No mention is made in the abstract as to what type of lipoxygenase the activity relates to. Nor is there a mention as to a reduction in hexanal production and/or its odor or flavor.
Japanese Patent Application JA-033447 to Ajinomoto KK describes the production of a cheese-like protein food obtained by treating soybean with a mixture of enzymes to improve its taste and flavor. One of the enzymes utilized is lipase which renders a product free of bean smell. However, no description of the enzyme is made in the abstract.
Japanese Patent Application JP-052815 relates to the production of soybean protein by treating soybeans having the activities of two of the three lipoxygenase isoenzymes L-1, L-2 and L-3 simultaneously eliminated. This reference teaches away from the present invention.
Japanese Patent Application JP-170701 to Takasago Perfumery Kk relates to the preparation of a grass-flavored substance by grinding raw soybeans, adding unsaturated fatty acids, and optionally lipase enzyme, and stirring. It is said in the abstract that the yield of flavored substance can be increased by utilizing the lipase enzyme.
Japanese Patent Application JP 154721 relates to a food composition containing proteins decomposed by enzymes such as lipase. Among the foods is listed soybean juice, of which it is said that a more digestible form with high nutritive value is obtained.
U.S. Pat. No. 4,769,243 to Kanisawa et al. is related to JP-170701 discussed above and discloses a method of preparing compounds with green aroma by grinding raw soybeans in water at or below 60.degree. C., in the presence of air or oxygen. A lipase enzyme can be optionally added or substituted for the unsaturated higher fatty acids. A stronger green aroma is said to be obtained by adding both the fatty acid and the lipase together (see column 2, lines 1-8, 51-69 of Kanisawa et al.). The patent indicates that n-hexanal is utilized in the production of green aroma (see column 1, lines 21-24 of Kanisawa).
U.S. Pat. No. 4,232,044 to Chiba et al. describes the improvement of food protein flavor by the enzymatic conversion of aldehydes and alcohols (see column 2, lines 26-42 of Chiba et al.) by utilizing an aldehyde dehydrogenase and an alcohol dehydrogenase. Also disclosed is a process for improving the flavor of protein containing aldehydes and alcohols which relies on the reaction of the protein with aldehyde dehydrogenase or aldehyde oxidase to convert aldehydes to acids (see e.g., claim 1 of the patent).
U.S. Pat. No. 3,718,479 relates to a treatment of soybeans for use in processed foods involving the addition of sulfurous acid and lactic acid bacteria, steaming and fermentation with proteolytic and macerating activity containing microorganisms, drying and pulverizing the soybeans. The process is particularly interesting because enzymes such as lipoxygenase are inactivated. This teaches away from the present invention (see column 2, lines 38-41 of Kanno et al.).
U.S. Pat. No. 3,585,047 to Fujimaki et al. relates to a process of incubating soybean curd or defatted soybean flour with proteolytic enzymes to release a beany and astringent flavor and lipid materials from the proteinaceous constituents. The process is designed for removing among others the hexanal content of the product (see column 2, lines 20-47 of Fujimaki et al.) by means of enzymes such as proteolytic enzymes.
U.S. Pat. No. 3,048,492 to Barton relates to a reduction in the flavor of leguminous food materials by treating with an oxidizing enzyme to elevate the carbonyl content thereof (see column 1, lines 16-22 of Barton). A series of leguminous foods are listed in column 2, lines 11-17 of this patent.
U.S. Pat. No. 4,642,236 to Friend et al. relates to a process for reducing undesirable flavor components in a vegetable protein material such as soybean derivatives by contacting with a mold of the genus Rhizopus or Asoerqillus. A source of the undesirable flavor is admitted to be the oxidation of native lipids contained in the soybean material. This patent attributes this undesirable flavor to the presence of lipoxygenase which can catalyze the oxidation of lipids and produce hydroperoxide compounds which can undergo further transformation by enzymatic and non-enzymatic means to yield products which also adversely affect the flavor of the soybean product. N-hexanal is mentioned as one of them (see column 2, lines 1-19 of Friend et al.)
U.S. Pat. No. 4,677,247 issued to Kitamura describes the production of odorless soybean products by breeding soybeans to produce a variety holding the homozygous recessive 1x.sub.2 and 1x.sub.3 genes which results in the lipoxygenase L-2 and L-3 lacking characteristics. Accordingly, the inventor proposes that the reduction in the level of enzyme lipoxygenase 3 will reduce the undesirable taste characterized by a grassy flavor and bitterness typical of soybean products. This is contrary to the present invention.
Accordingly, there is a need for improved plant products like soybeans and soybean flour and meal and wheat germ which have reduced amounts of undesirable odor producing ingredients. These products may be utilized in higher amounts than is currently possible with alimentary products.